Receptor tyrosine kinases (RTKs) are important in the transmission of biochemical signals across the plasma membrane of cells. These transmembrane molecules characteristically consist of an extracellular ligand-binding domain connected through a segment in the plasma membrane to an intracellular tyrosine kinase domain.
The human epidermal growth factor receptor (HER) family consists of four distinct receptor tyrosine kinases referred to HER1, HER2, HER3, and HER4. These kinases are also referred to as erbB1, erbB2, etc. HER1 is also commonly referred to as the epidermal growth factor (EGF) receptor. With the exception of HER3, these receptors have intrinsic protein kinase activity that is specific for tyrosine residues of phosphoacceptor proteins. The HER kinases are expressed in most epithelial cells as well as tumor cells of epithelial origin. They are also often expressed in tumor cells of mesenchymal origin such as sarcomas or rhabdomyosarcomas. RTKs such as HER1 and HER2 are involved in cell proliferation and are associated with diseases such as psoriasis and cancer. Disruption of signal transduction by inhibition of these kinases would have an antiproliferative and therapeutic effect.
The enzymatic activity of receptor tyrosine kinases can be stimulated by either overexpression, or by ligand-mediated dimerization. The formation of homodimers as well as heterodimers has been demonstrated for the HER receptor family. An example of homodimerization is the dimerization of HER1 (EGF receptor) by one of the EGF family of ligands (which includes EGF, transforming growth factor alpha, betacellulin, heparin-binding EGF, and epiregulin). Heterodimerization among the four HER receptor kinases can be promoted by binding to members of the heregulin (also referred to neuregulin) family of ligands. Such heterodimerization as involving HER2 and HER3, or a HER3/HER4 combination, results in a significant stimulation of the tyrosine kinase activity of the receptor dimers even though one of the receptors (HER3) is enzymatically inert. The kinase activity of HER2 has been shown to be activated also by virtue of overexpression of the receptor alone in a variety of cell types. Activation of receptor homodimers and heterodimers results in phosphorylation of tyrosine residues on the receptors and on other intracellular proteins. This is followed by the activation of intracellular signaling pathways such as those involving the microtubule associated protein kinase (MAP kinase ) and the phosphatidylinositol 3-kinase (PI3 kinase). Activation of these pathways have been shown to lead to cell proliferation and the inhibition of apoptosis. Inhibition of HER kinase signaling has been shown to inhibit cell proliferation and survival.
All protein kinases contain a structurally conserved catalytic domain of approximately 250–300 amino acid residues1. FIG. 1 shows an X-ray structure of HER12 which encompasses the highly conserved features of all members of the protein kinase family. The protein kinase fold is separated into two subdomains, or lobes. The smaller N-terminal lobe, or N lobe, is composed of a five-stranded β sheet and one prominent α helix, called helix αC. The C lobe is larger and is predominantly helical. The two lobes are connected through a single polypeptide strand (the linker/hinge region), which acts as a hinge about which the two domains can rotate with respect to one other upon binding of ATP and/or substrate. ATP is bound in the deep cleft between the two lobes and sits beneath a highly conserved loop connecting strands β1 and β2. This phosphate binding loop, or P loop, contains a conserved glycine-rich sequence motif (GXGXφG) where φ is usually tyrosine or phenylalanine. The glycine residues allow the loop to approach the phosphates of ATP very closely and to coordinate them via backbone interactions. The conserved aromatic side chain caps the site of phosphate transfer. ATP is anchored to the enzyme via hydrogen bonds between its adenine moiety and the backbone atoms of the linker region, and the ribose ring to residues at the start of the C-terminal domain.
Optimal phosphotransfer requires the precise spatial arrangement of several catalytic residues that are absolutely conserved among all known kinases. Asp813 and Asn818 (HER1 numbering as given in reference 2 or numbered as Asp837 and Asn842 as found in REFSEQ: accession NM—005228) emanate from a highly conserved loop structure at the base of the active site, called the catalytic loop. Asp813 interacts with the attacking hydroxyl side chain of the substrate, while Asn818 engages in hydrogen bonding interactions that orient Asp813. Asn818 and another absolutely conserved catalytic residue, Asp831 (numbered as Asp855 as found in REFSEQ: accession NM—005228), are also required for the binding of two divalent metal cations involved in coordination of the triphosphate group.
Numerous structures of complexes with ATP, its analogs, or small-molecule inhibitors bound to different protein kinases have provided a clear description of the organization of the catalytic domain and the ATP-binding cleft and of the similarities and differences that exist within the binding region3. It is now clear that there are regions within the binding cleft that are not occupied by ATP, and that these show structural diversity between members of the kinase family. FIG. 2 shows the interactions of ATP with the hinge region of human cyclin-dependent kinase 2 (CDK2)4. The generic regions of all known kinase ATP binding sites are delineated in the figure as: (1) the adenine binding region; (2) the ribose pocket; (3) the phosphate binding pocket; (4) a mostly hydrophobic region 1, behind the adenine ring, and (5) region 2, a cleft or a tunnel adjacent to the ribose pocket and the N3 nitrogen of adenine which points towards a surface-exposed area of the kinase domain. The available structures of kinase/inhibitor complexes indicate that one can take advantage of the regions not occupied by ATP, e.g. regions 1 and 2, for increasing binding interactions and hence binding potency and potentially because of sequence differences between kinases in these regions also modulate selectivity.
A combination of crystallography, modeling, screening and medicinal chemistry efforts has led to the understanding of the binding mode of the pyrrolotriazine chemotype in the ATP binding site. Based on an X-ray crystal structure of the pyrrolotriazine chemotype inhibitor in VEGFR-2, it has been shown that the pyrrolotriazine ring binds in the adenine pocket and makes several key interactions with the hinge region similarly to ATP. In this binding mode, the C5 group is directed into the highly conserved ribose-phosphate pockets. The C4 group, depending on its chemical constituency, can be directed into the specificity region 1 and the C6 group is directed into the specificity region 2. Modeling of enumerated examples of this chemotype in HER1 shows that the C5 group claimed in this invention can at the least occupy the ribose-phosphate pocket and interact with at least one or more of the absolutely conserved residues involved in phosphate binding, e.g., Asn818 and Asp831 (HER1 numbering).
The conserved nature of the kinase catalytic core structure makes it an excellent target for the generic kinase inhibitor template afforded by the pyrrolotriazine ring and the C5 group. This template can be successfully derivatized to make specific and potent kinase ATP-competitive inhibitors by targeting the poorly conserved areas of the ATP-binding site.
It has surprisingly been found that compounds of the invention and other compounds such as those disclosed in U.S. Pat. Nos. 5,457,105, 5,616,582 and 5,770,599, which contain a small aniline derivative as the substituent off of the C4 position of the bicyclic ring, exhibit both HER1 and HER2 activity.